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Title Neuropeptide Y activates phosphorylation of ERK and STAT3 in stromal vascular cells from brown adipose tissue, butfails to affect thermogenic function of brown adipocytes

Author(s) Shimada, Kohei; Ohno, Yuta; Okamatsu-Ogura, Yuko; Suzuki, Masahiro; Kamikawa, Akihiro; Terao, Akira; Kimura,Kazuhiro

Citation Peptides, 34(2), 336-342https://doi.org/10.1016/j.peptides.2012.02.012

Issue Date 2012-04

Doc URL http://hdl.handle.net/2115/49225

Type article (author version)

File Information Pep34-2_336-342.pdf

Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP

1

Neuropeptide Y activates phosphorylation of ERK and STAT3 in stromal vascular

cells from brown adipose tissue, but fails to affect thermogenic function of brown

adipocytes

Kohei Shimada1, Yuta Ohno

1, Yuko Okamatsu-Ogura

1, Masahiro Suzuki

1, Akihiro

Kamikawa1,2

, Akira Terao1, and Kazuhiro Kimura

1

1Department of Biomedical Sciences, Graduate School of Veterinary Medicine,

Hokkaido University, Sapporo, Japan

2Department of Basic Veterinary Medicine, Obihiro University of Agriculture and

Veterinary Medicine, Obihiro, Hokkaido, Japan

Running title: NPY in BAT

Corresponding author: Kazuhiro Kimura, Ph.D.

Department of Biomedical Sciences, Graduate School of Veterinary Medicine,

Hokkaido University, Sapporo 060-0818, Japan

Tel. & Fax: +81-11-757-0703, E-mail: k-kimura@vetmed.hokudai.ac.jp

2

Abstract

The thermogenic function of brown adipose tissue (BAT) is increased by

norepinephrine (NE) released from sympathetic nerve endings, but the roles of NPY

released along with NE are poorly elucidated. Here, we examined effect of NPY on

basal and NE-enhanced thermogenesis in isolated brown adipocytes that express Y1 and

Y5 receptor mRNA. Treatment of cells with NPY did not influence the basal and

NE-enhanced rates of oxygen consumption and cAMP accumulation. Treatment with

NPY also failed to induce ERK (Thr202/Tyr204) phosphorylation in the brown

adipocytes. In contrast, treatment with NPY increased ERK phosphorylation in cultured

stromal vascular cells from the BAT that express Y1 receptor mRNA. In the latter

treatment with NPY also increased STAT3 (Ser727) phosphorylation. These results

suggest that NPY mainly acts on stromal vascular cells in BAT and plays roles in the

regulation of their gene transcription through ERK and STAT3 pathways, while NPY

does not affect the thermogenic function of brown adipocytes.

Key words: BAT, norepinephrine, p44/p42 MAPK, NPY, STAT, thermogenesis

3

1. Introduction

Acute cold exposure and feeding significantly stimulate thermogenesis in the

brown adipose tissue (BAT) [6, 9, 25, 33, 35]. The sympathetic nervous system (SNS)

controls this adaptive thermogenesis through the activation of -adrenergic receptors

(ARs), especially 3AR. It is well established that ARs are coupled to the

stimulatory G-protein (Gs) and thereby sequentially activate adenylyl cyclase (cAMP

formation) and cAMP-dependent protein kinase (PKA). It is also established that active

PKA leads to activation of lipolytic enzymes and enhanced expression of the genes

encoding uncoupling protein-1 (UCP1). Furthermore, thermogenesis in the BAT is

principally dependent on the activation of UCP1, which facilitates proton leakage

across the inner mitochondrial membrane to dissipate the electrochemical gradient as

heat.

The co-existence of sympathetic and peptidergic innervation has been

demonstrated in BAT [5, 11, 23, 28, 38]. Among peptidergic neurotransmitters,

neuropeptide Y (NPY) levels in the BAT are significantly decreased and increased by

surgical excision of the sympathetic nerves to the BAT and putting an animal into a cold

environment, respectively [30]. One study suggested the presence of two separate

subpopulations of autonomic nerves in the BAT, that is, one fiber containing both NE

and NPY innervates the vascular system, and the other fiber containing only NE

innervates the brown adipocytes [5, 6]. Similarly, it is suggested that NPY co-localizes

with tyrosine hydroxylase in noradrenergic neurons, but only in the perivascular

nerve fiber network [11]. However, other studies showed that NE- and NPY-positive

fibers were also found around parenchymal brown adipocytes in mice [38] and rats [23,

28].

4

NPY exhibits its physiological effects through at least four receptors known as Y1,

Y2, Y4, and Y5. It is well established that NPY receptors are coupled to the inhibitory

G-protein (Gi) and thereby antagonize -adrenergic agonist-induced activation of

adenylyl cyclase [27, 36]. Moreover, NPY potentiates NE actions via -adrenoceptor

[18, 36, 37], and the potentiation is attributed to the suppression of -adrenergic action

[18]. In the BAT, NPY is suggested to have a vasoconstrictor role at arteriovenous

anastomoses [40] and a suppressive role in BAT thermogenesis [30]. However, when

treated with NPY and NE in combination for 14 days, rats increase their minimal

oxygen consumption by approximately 38% compared with vehicle controls [2]. Similar

phenomena are observed in cold acclimated rats [3]. However, direct effects of NPY on

BAT thermogenesis have not been elucidated yet.

Therefore, in the present study, we first examined the effects of NPY on brown

adipocytes expressing Y1 and Y5 NPY receptors. We also examined the effects of NPY

on stromal vascular cells (SVC) expressing Y1 NPY receptor.

5

2. Materials and methods

2. 1. Materials

Dulbecco’s modified Eagle’s medium (DMEM) and collagenase were purchased from

Wako Pure Chemicals Co. (Osaka, Japan). Fatty acid-free bovine serum albumin (BSA)

and isobutylmethylxanthine (IBMX) were purchased from Sigma-Aldrich Fine

Chemicals (St. Louis, MO, USA). Fetal calf serum (FCS) was obtained from Trace

Scientific Ltd. (Melbourne, Australia). The following antibodies were purchased from

Cell Signaling Technology (Beverly, MA, USA): anti-phospho-specific ERK1/2

(p44/42MAPK, Thr-202/Tyr-204) antibody, anti-ERK1/2 antibody,

anti-phospho-specific STAT3 (Ser-727) antibody, anti-phospho-specific STAT3

(Tyr-705) antibody, and anti-STAT3 antibody.

2.2. Animals

Experimental procedures and animal care were carried out in accordance with the

Guidelines of Animal Care and Use from Hokkaido University, and were approved by

the University Committee for the Care and Use of Laboratory Animals. Male

8-week-old C57BL/6J mice were obtained from Nihon SLC (Shizuoka, Japan), housed

under specific pathogen-free conditions at 24°C with a 12 h:12 h light:dark cycle, and

given food and water ad libitum.

2.3. Isolation of brown adipocytes and stromal vascular cells (SVC)

Mice were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/Kg).

The whole body was perfused first with Ca2+

- and Mg2+

-free PBS and then with

Krebs-Ringer bicarbonate HEPES buffer (KRBH, pH 7.4) containing collagenase (1

6

mg/ml), through a cannula inserted into the left ventricle with a drain from the right

atrium in an open-chest procedure. Interscapular BAT was then rapidly removed and

placed in PBS. After careful removal of extraneous tissue, the BAT of two mice was

transferred in KRBH containing fatty acid–free BSA (10 mg/ml), 2.5 mM glucose, and

collagenase (1 mg/ml), minced well, and incubated for 30 minutes at 37 °C with

shaking at 90 cycles/min. The cell suspension was passed through a 200-m nylon filter

and the filtrate was centrifuged at 200 g for 1 min at room temperature. The floating

cells were collected as brown adipocytes, and washed 3 times with KRBH to eliminate

collagenase. The cells were diluted to a density of 3-5 × 105 cells/ml for BAT with the

assay buffer [KRBH containing fatty acid–free BSA (40 mg/ml) and 2.7 mM glucose].

The cell suspensions were kept for 1 h at room temperature before analysis. The

remainder after the collection of adipocytes was centrifuged at 1,200 g for 10 min at

room temperature. The supernatant was removed and the pellet was suspended and

cultured in DMEM containing 10% FCS in collagen-coated dishes (Iwaki-Asahi Techno

Glass, Chiba, Japan), and the media were changed every 2 days. Cells between the first

and second passages were used for the experiments as stromal vascular cells (SVC).

2.4. Measurement of oxygen consumption in isolated brown adipocytes

Oxygen consumption of isolated adipocytes was measured polarographically with a

Clark-style oxygen electrode in a water-jacketed Perspex chamber at 37 °C [29]. The

cell suspension (200 μl) was added to a magnetically stirred chamber set with a

thermostat at 37°C and was adjusted to a final volume of 1 ml with the assay buffer.

The chamber was closed, and the cells were incubated for 5 min to determine the basal

respiratory rate. NE and/or NPY were then injected with a Hamilton syringe through a

7

small hole in the cover of the chamber. Oxygen concentration in the chamber was

monitored for 15 min. Oxygen consumption rates were calculated using a computer

program (782 System, Strathkelvin Instruments, Glasgow, Scotland, UK).

2.5. cAMP accumulation in isolated brown adipocytes

Isolated adipocytes (3 x 105

cells) were incubated in 0.9 ml of the assay buffer

containing 0.5 mM IBMX (a phosphodiesterase inhibitor), and stimulated with NE (1

M) and/or NPY (0.1 M) for 10 min. HCl (0.1 ml, 1N) was then added and samples

were kept on ice for 10 min before centrifugation at 15,000×g for 15 min at 4 °C. The

cAMP content in the supernatant was then measured using a cyclic AMP Assay kit

(Enzo Life Sciences International, Inc., Plymouth Meeting, PA, USA), according to the

protocol provided.

2.6. ERK activation in isolated brown adipocytes

Isolated adipocytes (5 x 104 cells) were incubated in 0.1 ml KRBH, and stimulated with

NE (1 M) and/or NPY (0.1 M) for 10 min. HCl (10 µl, 1N) was then added and

samples were kept on ice for 10 min before adding chloroform/methanol (1:2, 0.5 ml).

The solution was centrifuged at 15,000×g for 1 min at 4 °C, and the resultant pellet was

washed once with chloroform/methanol and solubilized in a sample buffer for

SDS-PAGE.

2.7. Western blotting

Aliquots of cellular protein as described above (20 g) were separated by SDS-PAGE

and the proteins were transferred onto PVDF membranes (Immobilon; Millipore,

8

Bedford, MA, USA). The membranes were incubated in a blocking buffer [20 mM

Tris/HCl (pH7.5)/150 mM NaCl] containing 0.1% Tween 20 and 1% skimmed milk,

and then in the buffer containing a primary antibody. The bound primary antibody was

visualized using horseradish peroxidase-linked goat anti-rabbit IgG (Zymed

Laboratories, South San Francisco, CA, USA) and chemiluminescent HRP Substrate

(Millipore) according to the manufacturer’s instructions. The intensity of

chemiluminescence for the corresponding bands was analyzed using Image J, a

public-domain image-processing and analysis program.

2.8. Cell culture and treatments

SVC were cultured onto 100 mm dishes in DMEM supplemented with 10% fetal bovine

serum in 5% CO2 in humidified air at 37°C. After cells were grown to confluence, they

were cultured in a serum-free medium for 24 h to render them quiescent. The cells were

then treated with NPY and vehicle (PBS) for 10 min at 37°C. Subsequently, the cells

were washed once with buffer [50 mM Hepes (pH7.5), 150 mM NaCl, 5 mM EDTA, 10

mM sodium pyrophosphate, 2 mM NaVO3, protease inhibitor mixture (Complete;

Roche Diagnostics, GmbH, Germany)], then lysed with the buffer containing 1%

Nonidet P-40. The lysate was kept on ice for 30 min and centrifuged at 15,000g for 15

min at 4°C. The resulting supernatant was stored at -30°C. Proteins were determined by

the Lowry method using BSA as a standard [26]. When treated, PD98059 (an ERK

kinase inhibitor; EMD Biosciences, Inc., La Jolla, CA, USA) was added to the cultured

cells 2 h before NPY stimulation. Aliquots of the cell lysate were analyzed by Western

blotting.

9

2.9. Cell proliferation assay

SVC (5 × 103 cells) were cultured in 96-well plates with DMEM containing 1% FCS

and increasing concentrations of NPY for 72 h. Then, the number of cells was assessed

using Cell Counting Kit-8 (DOJINDO, Kumamoto, Japan).

2.10. mRNA analysis

Total cellular RNA was isolated from isolated adipocytes, cultured cells and mouse

tissues by the guanidine-isothiocyanate method using RNAiso reagent (Takara Bio,

Shiga, Japan). First, 2 µg of total RNA was mixed with DNaseI reaction buffer [DNaseI

(Roche), 40 mM Tris-HCl (pH7.2), 6 mM MgCl2, ribonuclease inhibitor (Takara)]. The

reaction was carried out at 37 °C for 30 min, and terminated at 95 °C for 5 min. Then,

single-strand cDNA was synthesized using 100 units of Moloney murine leukemia virus

reverse transcriptase (Invitrogen Co., Carlsbad, CA, USA), 50 pmol poly(dT) primer,

and 20 nmol dNTP in a total volume of 20 µl at 37 °C for 1 h. The reaction was

terminated at 95 °C for 5 min.

The expression of mRNA was analyzed by conventional PCR or real-time PCR using

the cDNA as a template. PCR amplification was performed with 2.5 units Taq

polymerase (Ampliqon, Herlev, Denmark), 3 mM MgCl2, and 50 pmol forward and

reverse primers specific to the respective genes in a total volume of 25 µl. Denaturation

and annealing were performed at 94 °C and 58 °C for 30 sec, respectively, while

extension was performed at 72 °C for 60 sec. The primers used are summarized in Table

1. The PCR products were analyzed by electrophoresis in 2% agarose gel and stained

with ethidium bromide. The PCR products were subcloned into the pGEM-T Easy

10

vector (Promega, Madison, WI, USA). After the nucleotide sequence of each cDNA was

confirmed, the cDNA were used as standards for real-time PCR. To quantify Egr-1 gene

expression levels, real-time PCR was performed with a fluorescence thermal cycler

(Light Cycler System, Roche) using 0.5 mM of each primer (Table 1). The fluorescence

of SYBR Green (Qiagen, Hilden, Germany) at 530 nm was recorded at the end of the

extension phase and analyzed using the Light Cycler Software (Version 3). The level of

mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh)

mRNA was also

determined as an internal control.

2.11. Statistical analysis

The results are expressed as means SD. Statistical analyses were performed using

ANOVA and Bonferroni’s test, with p < 0.05 being considered statistically significant.

11

3. Results

Brown adipocytes and stromal vascular cells (SVC) were isolated from the

mouse interscapular BAT. RT-PCR analyses revealed that the former expressed Y1 and

Y5, but not Y2, types of NPY receptors, while the latter mainly expressed Y1 NPY

receptor (Fig. 1). To evaluate the effects of NPY on brown adipocytes, we measured in

vitro oxygen consumption rate of isolated adipocytes as an indicator of thermogenic

function. Oxygen consumption rate was 2.9 ± 0.7 nmol O2/min/105 cells before

stimulation and increased to 15.0 ± 4.3 nmol O2/min/105 cells after stimulation with NE

at 1 M and 10 M (Fig. 2A). When stimulated with NPY (0.01-1 M), oxygen

consumption rate did not change from the basal rate (Fig. 2B). When simultaneously

stimulated with NPY (0.1 M) and NE (1 M), oxygen consumption rate did not

change from the NE-enhanced rate (Fig. 2C). NPY also failed to modify the

NE-enhanced rate even at the sub-maximal concentration of NE used (0.1 M, Fig. 2D).

Next, we measured cAMP formation of isolated adipocytes in the presence of a

phosphodiesterase inhibitor. cAMP accumulation was 15.9 ± 11.0 pmol/105 cells before

stimulation and increased to 125.8 ± 1.9 pmol/105 cells after stimulation with NE at 1

M (Fig. 3A). When stimulated with NPY (0.1 M) in the absence or presence of NE

stimulation, cAMP accumulation did not change from the basal or NE-increased level,

respectively (Fig. 3A). Furthermore, when examining NPY-induced ERK signaling

pathway as reported previously [14, 16, 19, 34], NPY failed to activate activity-related

site-specific phosphorylation of ERK (Thr202/Tyr204) in isolated adipocytes while NE

stimulated it (Fig. 3B). Therefore, it is unlikely that NPY controls basal and

NE-regulated thermogenesis, although mRNAs of NPY receptors were expressed.

To evaluate the effects of NPY on SVC, we examined whether NPY induced

12

ERK phosphorylation. As shown in Figure 4, treatment of cells with NPY (30 nM and

100 n) induced the phosphorylation, indicating that functional NPY receptor was

present in SVC. We further examined STAT3 phosphorylation at Ser727 and Tyr705, as

the serine residue of STAT3 is known to be a downstream target of the ERK pathway

[15, 17, 22, 24, 39]. NPY treatment at the concentration of 30 nM or higher induced the

Ser phosphorylation, but not the Tyr phosphorylation (Fig. 5). In time course analyses,

NPY induced ERK phosphorylation in 3 min after the stimulation, which ended at 30

min, while NPY induced and ceased STAT3 phosphorylation at Ser727 at 10 min and

30 min after the stimulation, respectively (Figs. 4 and 5). Prior treatment of PD98059,

an ERK kinase (MEK) inhibitor, suppressed both phosphorylation of ERK and STAT3

at Ser727 (Fig. 6). Moreover, treatment of SVC with NPY increased the immediate

early gene, Egr-1 (early growth response 1, a STAT3-responsive gene) mRNA

expression at 1 h after the stimulation, and prior treatment of PD98059 suppressed the

expression (Fig. 7).

To evaluate the role of NPY in the regulation of SVC function, we examined

whether NPY induced cell growth since NPY promotes proliferation of adipocyte

precursor cells in visceral adipose tissue [42]. In contrast to the report, NPY failed to

stimulate cell growth of stromal vascular cells of BAT (Fig. 8).

13

Discussion

In the present study, we have demonstrated for the first time that NPY fails to

affect the basal and NE-enhanced cAMP production, ERK phosphoryllation and

oxygen consumption of brown adipocytes in vitro. These phenomena are inconsistent

with the previous findings that NPY has a suppressive role in the BAT thermogenesis

when given intraperitoneally [30]. As central activation and suppression of NPY

neuron decrease and promote BAT function, respectively [6, 8, 21], this inconsistency

may be explained by the assumption that intraperitoneally administered NPY leads to

activation of central NPY neurons. However, central action induced by peripheral NPY

remains to be elucidated. Our observation also conflicts with the reports showing that

NPY antagonizes -adrenergic agonist-induced activation of adenylyl cyclase [27, 36].

As mRNAs of Y1 and Y5 receptors coupled to Gi protein were detected in brown

adipocytes, suppression of cAMP production and prevention of increase in oxygen

consumption were simply expected. However, the failure of NPY to modulate the

cAMP production, oxygen consumption and also ERK activation suggests that

functional NPY receptors are lacking in brown adipocytes. Therefore, it is unlikely that

NPY directly controls basal and NE-regulated thermogenesis in a short period of time,

even if some sympathetic nerves carrying both NE and NPY innervate brown

adipocytes [23, 28, 38].

In contrast to brown adipocytes, we have demonstrated that NPY activates ERK

phosphorylation in stromal vascular cells (SVC) from the BAT. NPY has been shown to

activate ERK pathway in a variety of cells, possibly leading to cell proliferation of

neuronal precursor cells [14, 16] and progression of cancer [34]. Moreover, Yang et al.

show that NPY promotes ERK-dependent proliferation of adipocyte precursor cells

14

from the white adipose tissue [42]. We also observed NPY induction of Egr-1 gene, a

transcription factor involved in cell proliferation and in the regulation of apoptosis [12,

13]. It is thus assumed that NPY released from sympathetic nerve terminal stimulates

proliferation of SVC, resulting in hyperplasia of the BAT, which occurs in response to

physiological stimuli such as cold exposure [20]. However, NPY failed to promote

proliferation of SVC from the BAT. The difference in cellular response to NPY in

adipocyte precursor cells between the white and brown adipose tissues may be simply

due to the different characteristics of these cells which expressing different genes.

STAT3 is a transcription factor and activated upon phosphorylation of the tyrosine

residue at amino acid 705 by Janus-activated kinase (JAK) upon the stimulation of a

variety of cytokine receptors [4]. In cultured SVC, STAT3 at Tyr705 was constitutively

phosphorylated by unknown mechanisms. As transformation of fibroblasts by viral Src

induces constitutive activation of STAT3 at Tyr705 [4, 7], non-receptor tyrosine kinases

such as cellular Src (c-Src) might be involved in the constitutive activation of STAT3 in

the cells used. Under these conditions, we have demonstrated for the first time that NPY

induced phosphorylation of STAT3 at Ser727. It is likely that ERK is a STAT3 kinase,

since ERK activation preceded phosphorylation of STAT3 at Ser727 and the MEK

inhibitor abrogated the phosphorylation of both ERK and STAT3 in SVC, as reported

previously [15, 17, 22, 24, 39].

Tyrosine phosphorylation of STAT3 plays pivotal roles in its nuclear translocation

and transcriptional activating function [4, 31], but it is a fact that the Y705F mutant of

STAT3, which cannot be phosphorylated on tyrosine, also activates some gene

expression [41]. There are also conflicting reports regarding the significance of the

serine phosphorylation of STAT3 in its transcriptional activating function [10]. That is,

15

STAT3 phosphorylation at Ser727 has been shown to reduce and increase the

transcriptional potential of STAT3. In either case, our result that NPY enhanced a

STAT3-responsible gene (Egr-1) expression in a MEK-dependent manner suggests the

possibility that NPY modulates ERK- and STAT3-dependent transcription via its Ser727

phosphorylation. In addition, NPY is known to control various genes including

neuropeptide precursors [1, 32]. Therefore, STAT3 may be important in NPY-dependent

transcriptional regulation.

In summary, the present results suggest that NPY acts on SVC in BAT and plays

roles in the regulation of their gene transcription through ERK and STAT3 pathways,

while NPY does not affect the thermogenic function of brown adipocytes.

16

Acknowledgements

This work was supported by grants from the Japan Society for the Promotion of Science

(JSPS) to K.K. The work was also supported by JSPS Research Fellowships for Young

Scientists to K.S.

17

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22

Table 1. Primer list

Gene Forward primer sequence (Accession No.) Reverse primer sequence

(PCR product size)

Npy1r 5`- TCA ACA GAG GTG AAC AGA CG -3` (NM_010934) 5`- TCA ATG CCA GGT TTC CAG AG -3`

(+69 to + 429, 361 bps) Npy2r 5`- ATG GGC CAG GGC ACA CTA CT -3` (NM_008731) 5`- GGG TGT TCA CCA AAA GAT CC -3`

(+339 to + 581, 243 bps)

Npy5r 5`- CAT GCC ATT CCT TCA GTG TG -3` (NM_016708) 5`- TCT GGA ACG GTT AGG TGC TT -3`

(+550 to + 1140, 591 bps)

egr1 5'- TGG CCT GAA CCC CTT TTC AG -3' (NM_007913) 5'- GTA GAT GGG ACT GCT GTC GT -3'

(+721 to + 885, 165 bps)

Gapdh 5'- GAA GGT CGG TGT GAA CGG ATT -3' (NM_008084) 5'- AAG ACA CCA GTA GAC TCC ACG A -3'

(+56 to + 349, 294 bps)

23

Figure legends

Fig. 1 Expression of NPY receptors.

Conventional RT-PCR was performed on the mRNA obtained from mouse brain,

stromal vascular cells (SVC) and brown adipocytes (BA) isolated from mouse

interscapular BAT in order to detect expression of NPY receptors, Y1, Y2, and Y5.

Fig. 2 Norepinephrine (NE) increased oxygen consumption of isolated brown

adipocytes, but NPY failed to modify basal and NE-enhanced oxygen consumption.

Oxygen consumption by brown adipocytes was monitored in vitro for 20 min, and

increasing concentrations of NE (A) and NPY (B) and their combination (C, D) were

added at 5 min after the onset of the monitoring (arrows). Data are mean ± SD of values

from 3 independent experiments.

Fig. 3 Norepinephrine (NE) increased cAMP formation and ERK phosphorylation

in isolated brown adipocytes, but NPY failed to modify basal and NE-induced

cAMP formation and ERK phosphorylation. Brown adipocytes were stimulated with

NE (1 µM) and/or NPY (100 nM) for 10 min in the presence (A) and absence (B) of a

phosphodiesterase inhibitor. Then, in A, cAMP levels were determined. In B,

immunoblot analyses were performed for total and phosphorylated ERK. Data are mean

± SD of values from 4 independent experiments in A and from 8 independent

experiments in B, respectively. *p<0.05 vs. without NE treatments.

Fig. 4 NPY increased phosphorylation of ERK in stromal vascular cells of BAT in

dose- and time-dependent manners. Cultured stromal vascular cells were stimulated

24

with increasing concentrations of NPY (0.1-100 nM) for 10 min in A and 100 nM NPY

for 3-60 min in B. Immunoblot analyses for total and phosphorylated ERK were

performed on 20 µg of protein of each sample, and representative blots are shown. Data

are mean ± SD of values from 3 independent experiments. *p<0.05 vs. control (0 nM or

0 min).

Fig. 5 NPY increased phosphorylation of STAT3 at Ser-727, but not Tyr705, in

stromal vascular cells of BAT in dose- and time-dependent manners. Cultured

stromal vascular cells were stimulated with increasing concentrations of NPY (0.1-100

nM) for 10 min in A and 100 nM NPY for 3-60 min in B. Immunoblot analyses for total

and phosphorylated STAT3 were performed on 20 µg of protein of each sample, and

representative blots are shown. Data are mean ± SD of values from 3 independent

experiments. *p<0.05 vs. control (0 nM or 0 min).

Fig. 6 PD98059 abrogated both NPY-induced phosphorylation of ERK and STAT3

at Ser727 in stromal vascular cells of BAT. Stromal vascular cells in culture were

stimulated with NPY (100 nM) for 10 min, in the presence of either DMSO (-) or

PD98059 (5-50 µM), an ERK kinase inhibitor. Immunoblot analyses for total and

phosphorylated ERK (A) and total and phosphorylated STAT3 (B) were performed on

20 µg of protein of each sample. Representative blots are shown. Data are mean ± SD of

values from 4 independent experiments. *p<0.05 vs. without NPY treatment. †p<0.05

vs. without PD98059 treatment.

Fig. 7 NPY increased Egr1 expression in stromal vascular cells of BAT, which was

25

inhibited by PD98059. Stromal vascular cells in culture were stimulated with NPY

(100 nM) for 1 h, in the presence of either DMSO (-) or PD98059 (+, 50 µM). The

mRNA expression of Egr-1, known as a STAT3-responsive gene, was visualized by

conventional RT-PCR and quantified by real-time PCR. Data are mean ± SD of values

from 3 independent experiments. *p<0.05 vs. without NPY treatment. †p<0.05 vs.

without PDD98059 treatment.

Fig. 8 NPY failed to stimulate cell growth of stromal vascular cells of BAT. Stromal

vascular cells (5 × 103 cells) were cultured in 96-well plates with DMEM containing 1%

FCS and increasing concentrations of NPY for 72 h. Then, the number of cells was

assessed and expressed as a relative value to the control cells without NPY treatment (0

nM). Data are mean ± SD of values from 7 independent experiments.